We performed SDS-PAGE and western blotting to detect the CREB5 and viral proteins. Total protein sample isolation and separation have been described elsewhere [25 (link)]. In brief, the cellular total proteins was boiled and separation in an 12% SDS-PAGE. The separated SDS-PAGE was then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA) and then washed. The membrane was and blocking with fast blocking solution (Biofuture Co., Taoyuan, Taipei) for 3 min prior to the primary and secondary antibodies incubation. The primary antibodies used were listed as the following: rabbit anti-EV-A71 VP (GeneTex, 1:1000), anti-Flag (Sigma, 1:3000 dilution) and anti-EV-A71 3D (1:5000 dilution); for detection of CREB5 (GeneTex, 1:2000) and β-actin (Sigma, 1:10000). The secondary antibodies were purchased from – (a) Goat anti-rabbit HRP secondary antibody (Abcam ab205718); (b) Goat anti-rabbit HRP secondary antibody (Abcam ab205719). The primary and secondary antibodies were diluted in the Super antibody mate solution (MDBio Co., Ltd., Taipei, Taiwan) followed by incubation ECL (BioMan biotech., Taipei, Taiwan) with the membranes. The signals of interested were detected using the ImageQuant™ LAS 4000 biomolecular imager (GE, USA).
Goat anti rabbit hrp secondary antibody
Manufactured by Abcam
Sourced in United States
Goat anti-rabbit HRP secondary antibody is a laboratory reagent used in various immunoassay techniques. It consists of goat-derived antibodies that specifically bind to rabbit primary antibodies, with a horseradish peroxidase (HRP) enzyme conjugated to facilitate signal detection. This secondary antibody can be used to amplify and visualize the signal from rabbit-based primary antibodies in applications such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ab205719, by Abcam (1 mentions)
Anti ev a71 3d, by GeneTex (1 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Anti flag, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Ecl advance western blotting detection kit, by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Rotenone, by Solarbio (1 mentions)
Citric acid, by Merck Group (1 mentions)
Anti egfr, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Anti vegfr2, by Abcam (1 mentions)
Anti p egfr, by Abcam (1 mentions)
Anti p akt, by Abcam (1 mentions)
Anti p vegfr2, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
8 protocols using goat anti rabbit hrp secondary antibody
1
Western Blot Detection of CREB5 and Viral Proteins
2
Antibody Storage and Usage Protocol
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Anti-HPRT mouse monoclonal antibody (MA5-15274) used for flow cytometry was aliquoted and stored at − 20 °C (Thermo Fischer Scientific, Waltham, MA, USA). Anti-HPRT rabbit polyclonal antibody (ab10479) used for Western blot analysis were purchased from Abcam (Cambridge, United Kingdom) and stored at 4 °C. Anti-Mouse-FITC and anti-Rabbit-FITC antibodies (Sigma Aldrich, St. Louis, MO, USA) were stored at 4 °C and were used in dark conditions. Goat-anti-rabbit-HRP secondary antibody was purchased from Abcam and stored at 4 °C.
3
Vimentin Staining for FLS Purity
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The SM tissue required for separated FLSs was labeled Vimentin to determine the purity of FLSs in SM. After the SM sections, antigen repair was undergone and then incubation with the anti-Vimentin primary antibody (Abcam, Cambridge, MA, USA), and goat anti-rabbit HRP secondary antibody (Abcam, Cambridge, MA, USA) was introduced for the final detection. The nuclei were counterstained using hematoxylin (Servicebio Tech., Wuhan, China) and sealed. The positive FLS expression was observed using an inverted fluorescence microscope.
4
Immunohistochemical Analysis of Cyclin D1
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Tissue was paraffin-embedded and sliced into 4 μm thick sections. Next, sections were paraffinized, rehydrated, and boiled at 100°C for 20 minutes to antigen retrieval. The sections were incubated with rabbit anti-human monoclonal cyclin D1 antibody (1 : 1000, Abcam) overnight at 4°C and goat anti-rabbit HRP secondary antibody (1 : 500, Abcam) 37°C for 30 minutes. Finally, the sections were stained with 3,3′-diaminobenzidine and then counterstained with hematoxylin.
5
Western Blot Analysis of Mitochondrial Proteins
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Total protein was extracted with RIPA lysis buffer containing 1% protease inhibitor cocktail (Abcam). Twenty micrograms of protein from mitochondria or whole cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were separated on a 10% separating gel and 3% stacking gel in the presence of 0.1% SDS and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Blocking for 1 hr was at room temperature with 5% non-fat milk. The membrane was incubated overnight with the primary antibodies (dilution ratio 1:1000) at 4°C with at room temperature, including EDNRB, COXIV and caspase-9 at a 1:1000 dilution in non-fat milk in TBS-T overnight at 4°C. The membrane was then washed 4 times in TBS-T and incubated with HRP-Goat Anti-rabbit secondary antibody (Abcam) at a 1:7500 dilution in non-fat milk in TBS-T for 1 hr at 37°C. The membrane was reacted with enhanced chemiluminescent reagents (ECL plus, GE Healthcare) and bands were visualized by an ECL Advance Western Blotting Detection Kit (Amersham).
6
Mitochondrial Respiration Assay
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Dimethyl malonate (dme), streptozocin, citric acid and sodium citrate were purchased from Merck KGaA. Rabbit SDHA antibody (cat. no. EPR9043) was purchased from Abcam. Rabbit GAPDH antibody (cat. no. EPR16891) was purchased from Abcam. HRP Goat anti-rabbit secondary antibody was purchased from Abcam (cat. no. ab205718). Reverse transcription kit SYBG-QPCR kit was purchased from Vazyme. The primers were purchased from Jiangsu Synthgene Biotechnology Co, Ltd.. Succinate acid, rotenone and adenosine diphosphate was purchased from Beijing Solarbio Science & Technology Co., Ltd..
7
Western Blot Analysis of Apoptosis and Signaling
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Cells were lysed using RIPA Lysis Buffer (Thermo Fisher Scientific) at 4°C. Proteins (30 μg per lane) were separated on 10% SDS-PAGE gel, and then transferred onto a polyvinylidene fluoride membrane (PVDF; Millipore, Billerica, MA, USA). Following this, the membrane was blocked with 5% non-fat milk for 1 hr at room temperature. Later on, the membrane was incubated with primary antibodies overnight at 4°C. Anti-cleaved caspase 3 (1:1000, Abcam Cambridge, MA, USA), anti-p-EGFR (1:1000, Abcam), anti-EGFR (1:1000, Abcam), anti-p-VEGFR2 (1:1000, Abcam), anti-VEGFR2 (1:1000, Abcam), anti-p-Akt (1:1000, Abcam), anti-Akt (1:1000, Abcam), anti-β-actin (1:1000, Abcam). After that, the membranes were incubated with HRP-goat anti-rabbit secondary antibody (1: 5000, Abcam) for 1 hr at room temperature. Enhanced chemiluminescence detection reagent (Tanon, Shanghai, China) was used to visualize the signal strength of the bands.
8
Immunohistochemical Analysis of EMT Markers
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Tissues were fixed in 10% formalin at 4 °C overnight and embedded in paraffin. Then, paraffin sections (4-μm thick) were permeabilized with 0.5% Triton X-100 in PBS for 1 h at room temperature, followed by incubation with specific primary antibodies at 4 °C overnight (vimentin, E-cadherin, STMN1). The sections were washed with PBS 3 times and then stained with HRP-conjugated secondary antibodies for 2 h at room temperature. Slides were imaged with an LSM510 confocal microscope system (Zeiss). The following primary antibodies were used: anti-vimentin (1:500; Abcam, USA), anti-E-cadherin (1:400; Abcam, USA), and anti-STMN1 (1:400; Abcam, USA). The following secondary antibodies were used: HRP goat anti-mouse secondary antibody (1:500, Abcam, USA) and HRP goat anti-rabbit secondary antibody (1:500, Abcam, USA).
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